The present invention relates to a method and apparatus for performing chemical and biochemical assays.
The ability to characterize processes at a cellular or sub-cellular level is important in both drug discovery and clinical diagnostics. One class of interactions frequently studied is the binding of one biological molecule to another molecule, cell or part of a cell. This may be for example the binding of antibodies to antigens, hormones to receptors, ligands to cell surface receptors, enzymes to substrates, nucleic acids to other nucleic acids, nucleic acids to proteins, and viruses to cell surfaces.
Another class of interactions important in the biology of the cell are diffusion or transport of molecules or cells across membranes. This may for example occur by osmosis; via special transport proteins or through phagocytosis.
Many diseases are characterised by binding or transport processes. In drug discovery the aim is to identify a means of enhancing or blocking the process. In clinical diagnostics the aim is to detect abnormal function of these processes; the presence of abnormal nucleic acid material; or to identify foreign bodies (such as viruses or bacteria) to diagnose a disease so that appropriate treatment may be given.
The present invention seeks to provide a rapid and simple assay to detect and quantify binding and transport processes important in drug discovery and clinical diagnostics.
For the purpose of the following discussion xe2x80x9creceptorxe2x80x9d shall mean any biological molecule, cell or structure that binds another molecule, cell or structure. Similarly xe2x80x9cligandxe2x80x9d shall mean any organic or inorganic molecule that binds to the xe2x80x9creceptorxe2x80x9d. The discussion and examples will focus on the assay of a labelled ligand binding to a receptor. The prior art described and the invention can be extended to include the interaction of a non-labelled agonist or antagonist in a competition assay as commonly used in drug discovery.
The basic principle of a reversible binding reaction is described by the equation:
Dissociation Constant (Kd)=[L]xc3x97[R]/[L.R ]
Where [L]=concentration of unbound ligand at equilibrium, [R]=concentration of unbound receptor at equilibrium, and [L.R]=concentration of bound ligand/receptor complex at equilibrium
The concentration is commonly measured in molar, and Kd for ligand; protein interactions is typically in the range 10xe2x88x924 to 10xe2x88x9215 Mxe2x88x921.
The classical assay used in drug discovery and diagnostics is the separation assay. In this assay one component (for example the ligand) is dissolved or suspended in solution. The other component (for example the receptor) may be immobilised to a surface such as the walls of a well in a microtitre plate, or may be present on the surface of a cell. One or both components may have a label such as a fluorescent or radioactive marker attached to it to assist measurement with an instrument. The assay is performed by adding the soluble component to a well containing the immobilised component and allowing the binding of the components to come to equilibrium. It is not possible with conventional detectors such as colourimetric, fluorescent or radioactivity plate readers to directly determine the amount of bound labelled ligand in the presence of free labelled ligand. This problem is overcome by separating the free ligand from the bound ligand by decanting off the solution containing the free ligand. One or more washes with fresh solvent may be performed to remove any excess free ligand. A measurement of the remaining label is assumed to represent the concentration of bound complex in the original solution. This process may also be performed where the receptor is on a cell. If the cells are not attached to the well the washing process is performed in special filter plates that retain the cells, but allow the wash solvent to pass through.
This method works well where the rate of dissociation of the bound complex is slow, and indeed it is used with success in many assays. However, there are significant disadvantages to this assay method when applied more widely.
If the rate of dissociation is fast some of the bound label will be released back into the wash solution resulting in an error during reading. The efficiency of washing itself may vary from one sample to the next, reducing the repeatability of the assay. It is desirable to reduce or eliminate washing steps in automated systems to increase throughput, reduce complexity and eliminate the risk of cross-contamination between samples.
A number of non-separation assay techniques have been developed in recent years to overcome these problems including Scintillation Proximity Assay (SPA), Fluorescence Polarisation (FP), Fluorescence Correlation Spectroscopy (FCS), and Time Resolved Fluorescence (TRF). However, each of these techniques has disadvantages that limit their applicability.
SPA relies on the transfer of energy from a radiolabelled ligand to a scintillant bead onto which the receptor is attached. The assay has to be conducted at relatively high concentrations to produce enough signal. Legislation on the disposal of radioactive material and the risk of exposure to operators has led to companies seeking alternatives. SPA is not suited to some assays using whole cells and cannot be used to assay receptors or proteins inside cells. This means that functional receptor must be isolated from the cell to perform the assay, and this is costly, difficult and in some cases cannot be achieved.
FP is a technique for estimating the mass of a fluorescent object from its speed of rotation or translocation through diffusion. The sample is illuminated by a burst of polarised light and emitted fluorescence is measured in the same or other polarisation plane. If the label is bound to a large object, rotation or translocation will be slower and emission will be in the same polarisation plane as the excitation for some time after the illumination. If the free label is much smaller than the bound complex the molecule may more rapidly move out of the plane of the incident polarised light and emit in another plane. Provided that the fluorophore has a sufficiently long decay time, the light reaching the detector will take longer to decay after excitation if a substantial number of fluorophore-labelled ligands in the solution are bound to larger molecules.
The method is a correlation rather than a direct measurement of bound to free label. Some of the free label will emit in the same plane as the excitation. It also requires that the labelled ligand be very much smaller than the receptor and that decay time for the fluorophore be longer than the speed of rotation of target molecules. This technique has many drawbacks: it is difficult to differentiate non-specific binding and contaminating background fluorescence from specifically bound labelled ligand; it cannot be used to study intracellular interactions; the sensitivity of the method is reduced by relying on the decay of the signal rather than peak fluorescence and it is limited to the use of certain fluorophores.
FCS is similar to FP with the exception that FCS performs correlations on single molecules. The technique predicts the size of a fluorescent particle or molecule from its speed of translocation through a fixed laser beam by brownian motion. To perform the technique it is desirable that only a single molecule of fluorophore be present in the laser beam at one time. There is a practical limit to how narrow the laser beam can be (typically of the order of a few microns in diameter). It is also impractical to have extremely short path lengths through the fluid. For this reason FCS is usually performed with very low concentrations of label. This technique is highly susceptible to contaminating background fluorescence typical in practical assays. It is also comparatively slow, taking up to half an hour of continuous measurement to detect binding to larger molecules.
The technique of FCS has been known for more than twenty years. The difficulties of using it for practical assays has prevented its use until comparatively recently. Some of the drawbacks of the technique are: fixed beam FCS examines only one interaction at a time which may not be representative of the whole sample; it cannot differentiate directly between specific and non-specific binding; the technique requires running assays at very low concentration, which can bring additional problems such as loss of signal through non-specific binding of the label or receptor to the walls of the vessel and low signal to noise ratio as a result of low signal strength. The technique is also susceptible to thermally induced eddy currents. These severe limitations could be reduced by employing an established technique used in the study of flow in liquids. By scanning the laser beam it would be possible to take a snapshot of the location of a number of fluorescent particles. Subsequent snapshots could determine the speed and direction of translocation of these particles and hence their mass. However, many of the fundamental limitations of the technique listed above would still apply.
TRF is similar to SPA in that it relies on the transfer of energy from one molecule to another in close proximity. In this case energy from one fluorophore is transferred to another fluorophore in close proximity. The technique requires both the receptor and the ligand to be soluble and that a fluorophore be present on both the ligand and the receptor. This is not suitable for assays where a soluble receptor cannot be obtained, and in addition chemically modifying the receptor by the addition of a label can be difficult and lead to a reduction or elimination of activity.
Over the past several years a number of instruments and techniques have been introduced for low throughput screening of cells based on imaging techniques using microscope objectives and/or CCD cameras. Typical of these are flourescent microscopes and scanning CCD systems. These systems employ a light source to alluminate a clear-bottomed plate from below. Cells are grown or deposited in the bottom of the wells and a fluorescent label or reagent is added to the solution above the cells. The detector is focussed only on the bottom of the well (in the cell sheet) to avoid obtaining signals from the bulk solution containing free label. The sample is imaged onto a CCD array and the resulting frame analysed for brightness by software. This approach can be used to image fluorescence within or binding to cells or beads, but there are several drawbacks, which limit its use for quantitative assays.
A CCD has finite resolution. The largest CCDs available today have around 1 million pixels, but cost-effective devices used in scientific devices have significantly fewer. There is therefore a compromise between field of view and resolution. This means that typically the field of view is only 1 mm2 with resolution of 4 xcexcm at best. This is only sufficient to obtain poorly resolved images of around 100 cells at once, which is insufficient for obtaining statistically significant results in some assay types. The resolution is insufficient to allow accurate measurement of the size and shape characteristics of cells or beads. The sensitivity of CCDs is substantially less than PMTs, making them insufficiently sensitive for making quantitative measurements at low light levels (for example labelled ligand bound to cell surface receptors on cells where expression is low, perhaps only 5,000 receptors per cell). It is necessary for quantitative competition assays to be able to measure bound fluorescence well below saturation, and CCD imaging systems lack this ability. Each pixel of the CCD array has a different sensitivity, so measurements across the scan are not consistent. Each pixel can only detect one colour at a time. Multi-colour images may be obtained by using a filter wheel in front of the CCD array, and taking multiple frames with different filters. This slows down the reading time, and if the sample moves during measurement (as free cells and beads are likely to do in liquid) the spectral information is lost. Multiple CCD arrays can be used to collect images in multiple colours, but it is not possible to achieve perfect pixel alignment between detectors or to have true simultaneous multi-wavelength detection. CCD arrays do not exhibit uniform sensitivity across the visible range. It is very important for background rejection at low signal levels that true simultaneous spectral measurements are made.
CCD arrays are not capable of repeatedly scanning an area at rates fast enough to perform measurements of rapid transients or time resolved fluorescence techniques (nanosecond to microsecond sampling rates).
Some systems, such as the xe2x80x9cFLIPRxe2x80x9d from Molecular Devices and FMAT from Perkin Elmer scan the sample with a laser. These systems employ confocal optics to deliberately limit the depth of the field of the detector, thus minimising the background signal from free label. This signal is not used to measure bound:free label concentrations. Additionally, resolution of these systems is too poor to allow accurate measurement of the shape or size of small beads or cells.
All the imaging systems do not attempt to measure the concentration of free label. For assays resulting in a dynamic equilibrium such as receptor/ligand binding assays it is necessary to have a measurement of both the free and the bound ligand to calculate the equilibrium constant or related measurements such as IC50. This is particularly important in practical assays where variability in the liquid handling devices and the effect of evaporation can mean that the concentration of ligand in the assay does not correspond with the desired concentration.
In addition to the limitations described for each technique there are general drawbacks with all these techniques for application across the widest range of assays. With the development of high throughput screening it is important that as many types of assay as possible be carried out in a single system. Each of the above techniques needs its own detector. This often leads to screening teams having to bolt different detectors into their robotic systems when changing from one assay type to another. This involves down time, and typically also involves re-programming of the robotics.
Fluorescence detection is becoming the method of choice for drug discovery because it offers sensitivities approaching that of radio label assays without the health risks and disposal problems. The present embodiment, offers a practical solution to the problems discussed above.
According to the present invention there is provided a method of performing a non-separation assay for determining the level of binding of one component to another, the method comprising the steps of:
providing a first component in solution;
providing an array of sites onto or into which is placed a second concentrated component;
immersing the array of sites with the solution;
scanning the array of sites with an illuminating light beam such that the light passes through the solution whilst illuminating the sites;
determining the intensity of light received from each of the sites and solution at at least one wavelength during illumination; and
using the received light intensity to determine a reference value representative of the solution alone and a value indicative of the amount of binding of the first component to the second component.
Applying mathematical computation to the signals received by the detector enables certain parameters to be determined.
The illuminating light may be generated by a laser beam. The received light may be light generated by fluorescence, and more than one wavelength of light may be received.
The received intensity may be employed to determine the size and/or volume of each site and the number of molecules bound to the site. The site may be formed by any surface or particle onto or into which a component may be concentrated, e.g. a cell, bead patterned surface or simply by a well.
The illuminating light may be arranged to illuminate from above or below the sample with the emitted light being detected from above or below the sample in any combination in such a way that the illuminating light illuminates both the site and a significant volume of the solution above or adjacent to the site.
The present invention also provides an apparatus for performing the above method.
An advantage of the present invention is that it can be employed to provide a reference value to the solution in which the sites sit so that the concentration of any fluorescent component in the solution may be measured (free component) and the number of molecules of any fluorescent component bound to the site (bound component) may be measured and compensation can be made for any signal from the free component that is coincident with the signal from the bound component so that an accurate value for the number of molecules bound to the site may be measured, and further, bound:free ratios may be estimated without need to separate the components. Furthermore, it is possible with the method and apparatus of the invention to determine not only the amount of binding but the area/volume of each site to ensure more accurate results.
The light source may scan the solution and sites in a linear fashion, with one scan overlapping the next, so that a continuous measurement of received light intensity can be provided. Data relating to the received light intensity may be filtered by employment of a fixed or variable threshold in order to reduce the amount of data required to be processed.
A further advantage of the present invention is that, by employing continuous scanning of the sites and solutions it is possible to determine accurately site locations and also to provide a reliable indication of spurious results caused by contamination and the like.
Yet another advantage of the present invention is that the meniscus of the sample may be determined simultaneously with the measurement of bound: free and compensated for in the mathematical analysis.